Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.

High-resolution imaging has enabled scientists to explore the mitochondrial network at remarkable resolution. This has been exploited to help increase our knowledge of how mitochondrial gene expression is compartmentalized in cultured cells. Here, we provide detailed methodology to simultaneously visualize up to four components including mtDNA-encoded transcripts, submitochondrial marker proteins, mitoribosomal subunits, or core members of the translational apparatus using STED super-resolution nanoscopy.

Mitochondrial Translation Occurs Preferentially in the Peri-Nuclear Mitochondrial Network of Cultured Human Cells.

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation.

Visualizing Mitochondrial Ribosomal RNA and Mitochondrial Protein Synthesis in Human Cell Lines.

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear.

Mitochondrial transplantation-a possible therapeutic for mitochondrial dysfunction?: Mitochondrial transfer is a potential cure for many diseases but proof of efficacy and safety is still lacking.

Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?

Messenger RNA delivery to mitoribosomes - hints from a bacterial toxin.

In mammalian mitochondria, messenger RNA is processed and matured from large primary transcripts in structures known as RNA granules. The identity of the factors and process transferring the matured mRNA to the mitoribosome for translation is unclear. Nascent mature transcripts are believed to associate initially with the small mitoribosomal subunit prior to recruitment of the large subunit to form the translationally active monosome.

Rescuing stalled mammalian mitoribosomes - what can we learn from bacteria?

In the canonical process of translation, newly completed proteins escape from the ribosome following cleavage of the ester bond that anchors the polypeptide to the P-site tRNA, after which the ribosome can be recycled to initiate a new round of translation. Not all protein synthesis runs to completion as various factors can impede the progression of ribosomes. Rescuing of stalled ribosomes in mammalian mitochondria, however, does not share the same mechanisms that many bacteria use.

How much does a disrupted mitochondrial network influence neuronal dysfunction?

Mitochondria are organelles that are present in all nucleated cells in the body. They have manifold functions but famously generate ATP efficiently through the process of oxidative phosphorylation. This ensures all tissues have an adequate energy supply and underlines the need for a fully functional mitochondrial network.

mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect.

OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle.

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies.

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure.

The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome.

Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis.

Store-Operated Ca Entry Controls Induction of Lipolysis and the Transcriptional Reprogramming to Lipid Metabolism.

Ca signals were reported to control lipid homeostasis, but the Ca channels and pathways involved are largely unknown. Store-operated Ca entry (SOCE) is a ubiquitous Ca influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca channel ORAI1. We show that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle.

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules.

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking.

Human mitochondrial nucleases.

Mitochondria are cytosolic organelles that have many essential roles including ATP production via oxidative phosphorylation, apoptosis, iron-sulfur cluster biogenesis, heme and steroid synthesis, calcium homeostasis, and regulation of cellular redox state. One of the unique features of these organelles is the presence of an extrachromosomal mitochondrial genome (mtDNA), together with all the machinery required to replicate and transcribe mtDNA. The accurate maintenance of mitochondrial gene expression is essential for correct organellar metabolism, and is in part dependent on the levels of mtDNA and mtRNA, which are regulated by balancing synthesis against degradation.

Adult Onset Leigh Syndrome in the Intensive Care Setting: A Novel Presentation of a C12orf65 Related Mitochondrial Disease.

Background: Mitochondrial disease can present at any age, with dysfunction in almost any tissue making diagnosis a challenge. It can result from inherited or sporadic mutations in either the mitochondrial or the nuclear genome, many of which affect intraorganellar gene expression. The estimated prevalence of 1/4300 indicates these to be amongst the commonest inherited neuromuscular disorders, emphasising the importance of recognition of the diagnostic clinical features.

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence.

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress.

Human mitochondrial ribosomes can switch their structural RNA composition.

The recent developments in cryo-EM have revolutionized our access to previously refractory structures. In particular, such studies of mammalian mitoribosomes have confirmed the absence of any 5S rRNA species and revealed the unexpected presence of a mitochondrially encoded tRNA (mt-tRNA) that usurps this position. Although the cryo-EM structures resolved the conundrum of whether mammalian mitoribosomes contain a 5S rRNA, they introduced a new dilemma: Why do human and porcine mitoribosomes integrate contrasting mt-tRNAs? Human mitoribosomes have been shown to integrate mt-tRNA compared with the porcine use of mt-tRNA We have explored this observation further.

Pathogenic variants in HTRA2 cause an early-onset mitochondrial syndrome associated with 3-methylglutaconic aciduria.

Mitochondrial diseases collectively represent one of the most heterogeneous group of metabolic disorders. Symptoms can manifest at any age, presenting with isolated or multiple-organ involvement. Advances in next-generation sequencing strategies have greatly enhanced the diagnosis of patients with mitochondrial disease, particularly where a mitochondrial aetiology is strongly suspected yet OXPHOS activities in biopsied tissue samples appear normal.

The process of mammalian mitochondrial protein synthesis.

Oxidative phosphorylation (OXPHOS) is the mechanism whereby ATP, the major energy source for the cell, is produced by harnessing cellular respiration in the mitochondrion. This is facilitated by five multi-subunit complexes housed within the inner mitochondrial membrane. These complexes, with the exception of complex II, are of a dual genetic origin, requiring expression from nuclear and mitochondrial genes.

SLIRP stabilizes LRPPRC via an RRM-PPR protein interface.

LRPPRC is a protein that has attracted interest both for its role in post-transcriptional regulation of mitochondrial gene expression and more recently because numerous mutated variants have been characterized as causing severe infantile mitochondrial neurodegeneration. LRPPRC belongs to the pentatricopeptide repeat (PPR) protein family, originally defined by their RNA binding capacity, and forms a complex with SLIRP that harbours an RNA recognition motif (RRM) domain. We show here that LRPPRC displays a broad and strong RNA binding capacity in vitro in contrast to SLIRP that associates only weakly with RNA.

LRPPRC mutations cause early-onset multisystem mitochondrial disease outside of the French-Canadian population.

Mitochondrial Complex IV [cytochrome c oxidase (COX)] deficiency is one of the most common respiratory chain defects in humans. The clinical phenotypes associated with COX deficiency include liver disease, cardiomyopathy and Leigh syndrome, a neurodegenerative disorder characterized by bilateral high signal lesions in the brainstem and basal ganglia. COX deficiency can result from mutations affecting many different mitochondrial proteins.

The presence of highly disruptive 16S rRNA mutations in clinical samples indicates a wider role for mutations of the mitochondrial ribosome in human disease.

Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge.

A truncating PET100 variant causing fatal infantile lactic acidosis and isolated cytochrome c oxidase deficiency.

Isolated mitochondrial complex IV (cytochrome c oxidase) deficiency is an important cause of mitochondrial disease in children and adults. It is genetically heterogeneous, given that both mtDNA-encoded and nuclear-encoded gene products contribute to structural components and assembly factors. Pathogenic variants within these proteins are associated with clinical variability ranging from isolated organ involvement to multisystem disease presentations.